Neurotransmitter transporter

ABSTRACT

Disclosed is a neurotransmitter transporter protein and DNA (RNA) encoding such protein. Also provided is a procedure for producing such polypeptide by recombinant techniques. The procedure for producing antagonists/inhibitors against such polypeptide is also provided. Such antagonists/inhibitors may be used to inhibit the action of neurotransmitter transporter protein for treatment of depression, anxiety of epilepsy.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention is a neurotransmitter transporter and the polypeptide of thepresent invention is herein sometime referred to as "NTT". The inventionalso relates to inhibiting the action of such polypeptides.

An essential property of synaptic transmission is the rapid terminationof action following neurotransmitter release. For manyneurotransmitters, including catecholamines, serotonin, and certainamino acids (e.g., γ-aminobutyric acid (GABA), glutamate, and glycine),rapid termination of synaptic action is achieved by the uptake of thetransmitter into the presynaptic terminal and surrounding glial cells byneurotransmitter transporters (Bennett, et al., Life Sci. 15:1045-1056(1974)). Inhibition or stimulation of neurotransmitter uptake provides ameans for modulating the strength of the synaptic action by regulatingthe available levels of endogenous transmitters. Neurotransmittertransporters are membrane-bound polypeptides which uptakeneurotransmitters into the pre-synaptic neuron after theneurotransmitters have crossed the synaptic cleft and acted upon thepost-synaptic neuron. Neurotransmitters can be excitatory, such asglutamate, or inhibitory such as GABA.

Affinity neurotransmitter transport is thought to terminate the overallprocess of synaptic transmission (Iversen, L. L., Br. J. Pharmacol.41:571-591 (1971)). Recently, cDNAs encoding more than ten differentneurotransmitter transporters have been cloned and sequenced. The familyof these genes could be divided into three subfamilies, including theGABA and taurine transporters (Liu, Q. R., et al., Proc. Natl. Acad.Sci. USA (in press), (1992)), the amino acid (glycine and proline)transporters (Fremeau, Jr., R. T., et al., Neuron, 8:915-926 (1992)),and the catecholamine transporters (Pacholczyk, T., et al., Nature,350:350-354 (1991)). The general structure of all these gene products isvery similar. They contain twelve potential transmembrane helices and anextended external loop with 3-4 glycosylation sites between membranesegments 3 and 4. The calculated molecular weights of the transportersis about 70 kDa and both their C- and N-terminal peripheral peptidescontain about 40 amino acids and may be located on the cytoplasmic sideof the membrane. In GABA and catecholamine transporter subfamilies, theamino acid sequence of each member is 60-80% identical to the othermembers within a subfamily and about 40% identical to members betweenthe two subfamilies (Liu, Q. R., et al., Proc. Natl. Acad. Sci. USA,89:6639-6643 (1992)). Amino acid transporters, such as the glycinetransporter and proline transporter, share about 40-45% homology withall members of the neurotransmitter transporter superfamily. Sequencehomology among the members of the neurotransmitter transporter familygive clear indication that they evolved from a common ancestral gene.Moreover, partial genomic cloning of several neurotransmittertransporters reveal that in all of them the first intron in the readingframe is located in an identical position (id.).

A GABA_(A) transporter was the first neurotransmitter system to becloned and expressed (Guastella, J., et al., Science 249:1303-1306(1990)) and is one of a family of neurotransmitter transporters clonedwithin the last year. Recently, a serotonin transporter cDNA has beendisclosed in PCT WO 93/08261.

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is herein referred to as NTT,as well as fragments, analogs and derivatives thereof. The polypeptideof the present invention is of human origin.

In accordance with another aspect of the present invention, there areprovided polynucleotides (DNA or RNA) which encode such polypeptides.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques.

In accordance with yet a further aspect of the present invention, thereare provided agonists which increase the affinity of NTT for itssubstrate, and which may be used to treat Amyotrophic Lateral Sclerosis,pain and stroke.

In accordance with a further aspect of the present invention, there areprovided antibodies against such NTT polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonist/inhibitors which may be used to prevent theuptake of neurotransmitters by NTT, which may be used therapeutically,for example, in the treatment of depression, anxiety and epilepsy, aswell as other neurologic or psychiatric disorders.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims. (FIGS. 1A, 1B, 1C, 1D and 1E collectively show (FIG. 1Aillustrates the first portions of the polynucleotide sequence encodingthe mature NTT polypeptide cDNA sequence (SEQ ID NO:1) and itscorresponding amino acid sequence and FIGS. 1B-1E consecutively continuewith the second, third, fourth and fifth parts, respectively, to the endof the same polynucleotide and amino acid sequences). The standardone-letter abbreviations are utilized to represent the amino acidresidues in polypeptide sequence that is shown in FIGS. 1A-1E.

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1A-1E or forthe mature polypeptide encoded by the cDNA of the clone deposited asATCC Deposit No. 75713 on Mar. 18, 1994. This deposit is a biologicaldeposit with the ATCC, 12301 Parklawn Drive, Rockville, Md. 20852.

The polynucleotide of this invention was discovered in a cDNA libraryderived from a human fetal brain. It is structurally related to theneurotransmitter transporter family. It contains an open reading frameencoding a protein of about 727 amino acid residues. The proteinexhibits the highest degree of homology to a rat neurotransmittertransporter (NT74) with 94% identity and 96% similarity over the entireamino acid sequence.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1A-1E or that of the deposited cloneor may be a different coding sequence which coding sequence, as a resultof the redundancy or degeneracy of the genetic code, encodes the same,mature polypeptide as the DNA of FIG. 1A-1E or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIG.1A-1E or for the mature polypeptide encoded by the deposited cDNA mayinclude: only the coding sequence for the mature polypeptide; the codingsequence for the mature polypeptide and additional coding sequence suchas a leader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequence) and non-coding sequence, such as introns or non-codingsequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1A-1E or the polypeptide encoded by the cDNA of the depositedclone. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1A-2E or the same mature polypeptideencoded by the cDNA of the deposited clone as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the polypeptide of FIG. 1A-2E or the polypeptide encoded bythe cDNA of the deposited clone. Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1A-1E or of the coding sequence of the deposited clone. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexahistidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably 70% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term "stringent conditions" means hybridization will occur only ifthere is at least 95% and preferably at least 97% identity between thesequences. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNA of FIG. 1A-2E or thedeposited cDNA.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

The present invention further relates to an NTT polypeptide which hasthe deduced amino acid sequence of FIG. 1A-1E or which has the aminoacid sequence encoded by the deposited cDNA, as well as fragments,analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to thepolypeptide of FIG. 1A-1E or that encoded by the deposited cDNA, means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1A-1E orthat encoded by the deposited cDNA may be (i) one in which one or moreof the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term "isolated" means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the NTT genes. The culture conditions, suchas temperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker-genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila and Sf9;animal cells such as CHO, HEK 293, COS or Bowes melanoma; plant cells,etc. The selection of an appropriate host is deemed to be within thescope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by, for example, calcium phosphate transfection,DEAE-Dextran mediated transfection, or electroporation. (Davis, L.,Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 "backbone" sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HEK 293, HeLa andBHK cell lines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5' flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The NTT polypeptides can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The present invention also provides a method for identifyingneurotransmitters which interact with the NTT polypeptides of thepresent invention. The method for determining whether a neurotransmitteris translocated from the synaptic cleft into the pre-synaptic neuron byNTT comprises transfecting a cell population with the appropriate vectorexpressing the NTT such that the cell will now express NTT. Variousneurotransmitters are then radio-labelled, e.g., tritiated, andincubated with the transfected cell to identify which neurotransmittersare transported into the cell.

Once a neurotransmitter is identified compounds can be screened toidentify those which specifically interact with NTT and either increaseNTT's affinity to uptake its neurotransmitter, e.g., an agonist, ordecrease its ability to uptake a neurotransmitter, e.g., anantagonist/inhibitor. This method comprises transforming host cells witha vector of the present invention such that the NTT polypeptide isexpressed in that host, incubating the host cells with the naturalneurotransmitter of NTT which has been labelled by a detectable markersequence (e.g., radiolabel or a non-isotopic label such as biotin) andthe potential compound and determining whether translocation of theneurotransmitter into the cell is either inhibited or increased. Bymeasuring the amount of neurotransmitter inside the cell, one skilled inthe art could determine if the compound is an effective agonist orantagonist.

The presence of excitatory or inhibitory neurotransmitters haveimportant clinical significance. For example, glutamate is an excitatoryneurotransmitter and its presence in the synaptic cleft can be toxic toneurons. This neuronal toxicity has been found to play a significantrole in Amyotrophic Lateral Sclerosis or "ALS". Further, during a strokeexcessive concentrations of glutamate are released into the synapticcleft and are toxic to neuronal cells. Moreover, although the cause ofgeneral pain is unknown, it is believed that pain is characterized bythe release of neurotransmitters into the synaptic cleft in the brain.Accordingly, an agonist of NTT may be employed to stimulate the uptakeof neurotransmitters and therefore alleviate these above-mentionedconditions.

The NTT polypeptides of the present invention may be administered byexpression of such polypeptides in vivo, which is often referred to as"gene therapy." Gene therapy is similar to the application of an NTTagonist, however, in gene therapy a polynucleotide of the presentinvention is administered such that the cellular machinery of the hostexpresses the NTT of the present invention to facilitate uptake ofneurotransmitters where that is desired, for example in ALS, stroke andgeneral pain.

For example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being targeted to the neuronal cells of a patientwhere expression of NTT and translocation of neurotransmitters aredesired. Such methods are well-known in the art. For example, cells maybe engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

The present invention is also directed to antagonist/inhibitors of thepolypeptides of the present invention, in addition to those identifiedby utilizing the above-described screening method. Antagonists includean antibody against the NTT polypeptide or, in some cases, anoligonucleotide which bind to the NTT making it inaccessible to itsnatural neurotransmitter allowing the concentration of theneurotransmitter in the synaptic cleft to increase.

Inhibitors include antisense constructs prepared using antisensetechnology. Antisense technology can be used to control gene expressionthrough triple-helix formation or antisense DNA or RNA, both of whichmethods are based on binding of a polynucleotide to DNA or RNA. Forexample, the 5' coding portion of the polynucleotide sequence, whichencodes the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix -see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251: 1360 (1991)), therebypreventing transcription and the production of NTT. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the NTT (antisense--Okano, J. Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of NTT.

In these ways, the antagonist/inhibitors may be used to treatdepression, anxiety, epilepsy and other neurological and psychiatricdisorders. Defects in neurotransmitter transport systems result inincreased or decreased concentrations of neurotransmitter in thesynaptic cleft, resulting in improperly stimulated receptors. Forexample, it has been postulated that depression is associated withdecreased release of norepinephrine and/or serotonin in the brain.Therefore, inhibiting NTT from translocating its neurotransmitter intothe presynaptic neuron would allow these neurotransmitters to interactmore frequently with their receptors. Accordingly, administration of theantagonist/inhibitors may be employed to alleviate the conditionsmentioned above. The antagonist/inhibitors may be employed in acomposition with a pharmaceutically acceptable carrier.

The present invention also relates to an assay for identifying potentialantagonist/inhibitors specific to NTT. An example of such an assaycomprises preparing a synaptosomal preparation from the hypothalamus ofa mammal. Such a preparation is a "sealed" neuron where the end of theneuron is pinched off. The synaptosomal preparation is then incubatedwith tritiated neurotransmitter and a potential antagonist. The degreeof uptake of neurotransmitter is then measured to determine if theantagonist is effective.

The compounds, e.g., agonist or antagonist/inhibitor compounds, of thepresent invention, may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the polypeptide, and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

The pharmaceutical compositions may be administered in an effectiveamount to effectively increase the affinity of NTT for itsneurotransmitter or inhibit NTT from translocating its neurotransmitter,and thereby alleviate the abnormal conditions associated with excessconcentrations of neurotransmitter in the synaptic cleft orconcentrations of neurotransmitter which are too low, as the case maybe.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA isused to rapidly select primers that do not span more than one exon inthe genomic DNA, thus complicating the amplification process. Theseprimers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases; however, clones larger than 2,000 bp havea higher likelihood of binding to a unique chromosomal location withsufficient signal intensity for simple detection. FISH requires use ofthe clones from which the EST was derived, and the longer the better.For example, 2,000 bp is good, 4,000 is better, and more than 4,000 isprobably not necessary to get good results a reasonable percentage ofthe time. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that cDNA sequence. Ultimately, completesequencing of genes from several individuals is required to confirm thepresence of a mutation and to distinguish mutations from polymorphisms.

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980). "Oligonucleotides" refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5' phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase ("ligase")per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

EXAMPLE 1 Bacterial Expression and Purification of NTT

The DNA sequence encoding for NTT, ATCC # 75713 is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5' and sequencesof the processed NTT protein (minus the signal peptide sequence) and thevector sequences 3' to the NTT gene. Additional nucleotidescorresponding to NTT were added to the 5' and 3' sequences respectively.The 5' oligonucleotide primer has the sequenceGACTAAAGCTTGGCATCAATGCCGAAGAAC (SEQ ID NO:3) contains a Hind IIIrestriction enzyme site followed by 18 nucleotides of NTT codingsequence. The 3' sequence GAACTTCTAGAGCAGTGGTCACAGCTCAG (SEQ ID NO:4)contains complementary sequences to Xba I site and is followed by 18nucleotides of NTT sequence. The restriction enzyme sites correspond tothe restriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE-9encodes antibiotic resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 was then digested with Hind III and Xba I. The amplified sequenceswere ligated into pQE-9 and were inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture wasthen used to transform E. coli strain M15/rep 4 available from Qiagenunder the trademark M15/rep 4 by the procedure described in Sambrook, J.et al., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies were selected.Plasmid DNA was isolated and confirmed by restriction analysis. Clonescontaining the desired constructs were grown overnight (O/N) in liquidculture in LB media supplemented with both Amp (100 ug/ml) and Kan (25ug/ml). The O/N culture is used to inoculate a large culture at a ratioof 1:100 to 1:250. The cells were grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalactopyranoside") was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized NTT was purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography411:177-184 (1984). NTT was eluted from the column in 6 molar guanidineHCl pH 5.0 and for the purpose of renaturation adjusted to 3 molarguanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced)and 2 mmolar glutathione (oxidized). After incubation in this solutionfor 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2 Expression of Recombinant NTT in COS Cells

The expression of plasmid, NTT HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E. coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire NTT precursor and a HA tag fused in frameto its 3' end was cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag correspond to an epitope derived from the influenzahemagglutinin protein as previously described (I. Wilson, H. Niman, R.Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).The infusion of HA tag to our target protein allows easy detection ofthe recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding for NTT, ATCC # 75713, was constructed by PCRon the original EST cloned using two primers: the 5' primerGACTAAGATCTGCCACCATGCCGAAGAACAGCAAAGTG (SEQ ID NO:5) contains a Bgl IIsite followed by 21 nucleotides of NTT coding sequence starting from theinitiation codon; the 3' sequence GAACTGATATCGCAGTGGTCACAGCTCAG (SEQ IDNO:6) contains complementary sequences to EcoR V site, translation stopcodon, and the last 18 nucleotides of the NTT coding sequence.Therefore, the PCR product contains a Bgl II site, NTT coding sequencefollowed by a translation termination stop codon, and an EcoR V site.The. PCR amplified DNA fragment and the vector, pcDNAI/Amp, weredigested with Bgl II and EcoR V. The ligation mixture was transformedinto E. coli strain SURE (available from Stratagene Cloning Systems,11099 North Torrey Pines Road, La Jolla, Calif. 92037) the transformedculture was plated on ampicillin media plates and resistant colonieswere selected. Plasmid DNA was isolated from transformants and examinedby restriction analysis for the presence of the correct fragment. Forexpression of the recombinant NTT, COS cells were transfected with theexpression vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T.Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989)). The expression of the NTT HA protein was detected byradiolabelling and immunoprecipitation method. (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells were labelled for 8 hours with ³⁵ S-cysteine two dayspost transfection. Culture media were then collected and cells werelysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM Tris, pH 7.5). (Wilson, I. et al., Id. 37:767(1984)). Both cell lysate and culture media were precipitated with a HAspecific monoclonal antibody. Proteins precipitated were analyzed on 15%SDS-PAGE gels.

EXAMPLE 3 Expression Pattern of NTT in Human Tissue

Northern blot analysis was carried out to examine the levels ofexpression of NTT in human tissues. Total cellular RNA samples wereisolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023 SouthLoop East, Houston, Tex. 77033). About 10 μg of total RNA isolated fromeach human tissue specified was separated on 1% agarose gel and blottedonto a nylon filter. (Sambrook, Fritsch, and Maniatis, MolecularCloning, Cold Spring Harbor Press, (1989)). The labeling reaction wasdone according to the Stratagene Prime-It kit with 50 ng DNA fragment.The labeled DNA was purified with a Select-G-50 column. (5 Prime - 3Prime, Inc. 5603 Arapahoe Road, Boulder, Colo. 80303). The filter wasthen hybridized with radioactive labeled full length MIP-2 gene at1,000,000 cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDS overnight at 65° C.After wash twice at room temperature and twice at 60° C. with 0.5×SSC,0.1% SDS, the filter was then exposed at -70° C. overnight with anintensifying screen. The message RNA for NTT is abundant in brain.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2485 BASE PAIRS                                                   (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGGAGGCAGGGAGTGAGGAGCGAGCGGAGTCGCGTGCGCCGGCGCGAGCTCCGGGTCGCC60                CCAGCCCCAGCCGGGGGCCTGTGGCGGGGGAGGAGCTGTGCGTCCGCGACCCGTCGGGAT120               CGCAGCTGCTCGGCCGGAGTGCACGGGCCGAGTCTGCGCGACTACCCACGCGTGACAGGT180               CCCTGAATGAGAAGGAGCTGACAGCAGCTGAATTCCATCTTCTCTGTGTGCTGGGGAGCA240               GGGCTACACGGCCCAGGTGGCATCAATGCCGAAGAACAGCAAAGTGACCCAGCGTGAGCA300               CAGCAGTGAGCATGTCACTGAGTCCGTGGCCGACCTGCTGGCCCTCGAGGAGCCTGTGGA360               CTATAAGCAGAGTGTACTGAATGTGGCTGGTGAGGCAGGCGGCAAGCAGAAGGCGGTGGA420               GGAGGAGCTGGATGCAGAGGACCGGCCGGCCTGGAACAGTAAGCTGCAGTACATCCTGGC480               CCAGATTGGCTTCTCTGTGGGCCTCGGCAACATCTGGAGGTTCCCCTACCTGTGCCAGAA540               AAATGGAGGAGGTGCTTACCTGGTGCCCTACCTGGTGCTGCTGATCATCATCGGGATCCC600               CCTCTTCTTCCTGGAGCTGGCTGTGGGTCAGAGGATCCGCCGCGGAAGCATCGGTGTGTG660               GCACTATATATGTCCCCGCCTGGGGGGGATCGGCTTCTCCAGCTGCATAGTCTGTCTCTT720               TGTGGGGCTGTATTATAATGTGATCATCGGGTGGAGCATCTTCTATTTCTTCAAGTCCTT780               CCAGTACCCGCTGCCCTGGAGTGAATGTCCTGTCGTCAGGAATGGGAGCGTCGCAGTGGT840               GGAGGCAGAGTGTGAAAAGAGCTCAGCCACTACCTACTTCTGGTACCGAGAGGCTTTGGA900               CATCTCTGACTCCATCTCGGAGAGTGGGGGCCTCAACTGGAAGATGACCCTGTGCCTCCT960               CGTGGTCTGGAGCATCGGGGGGATGGCTGTCGGTAAGGGCATCCAGTCCTCGGGGAAGGT1020              GATGTATTTCAGCTCCCTCTTCCCCTACGTGGTGCTGGCCTGCTTCCTGGTCCGGGGGTT1080              GTTGTTGCGAGGGGCAGTTGATGGCATCCTACACATGTTCACTCCCAAGCTGGTCAAGAT1140              GCTGGACCCCCAGGTGTGGCGGGAGGTAGCTACCCAGGTCTTCTTTGGCTTGGGTCTGGG1200              CTTTGGTGGTGTCATTGTCTTCTCCAGTTACAATAAGCAGGACAACAACTGCCACTTCGA1260              TGGCGCCCTGGTGTCCTTCATCAACTTCTTCACGTCAGTGTTGGCCACCCTCGTGGTGTT1320              TGTTGTTTTGGGCTTCAAGGCCAACATCATGAATGAGAAGTGTGTGGTCGAGAATGCTGA1380              GAAAATCCTAGGGTACCTTAACACCAACGTCCTGAGCCGGGACCTCATCCCACCCCACGT1440              CAACTTCTCCCACCTGACCACAAAGGACTACATGGAGATGGACAATGTCATCATGACCGT1500              GAAGGAGGACCAGTTCTCAGCCCTGGGCCTTGACCCCTGCCTTCTGGAGGACGAGCTGGA1560              CAAGTCCGTGCAGGGCACAGGCCTGGCCTTCATCGCCTTCACTGAGGCCATGACGCACTT1620              CCCCACCTCCCCGTTCTGGTCCGTCATGTTCTTCTTGATGCTTATCAACCTGGGCCTGGG1680              CAGCATGATCGGGACCATGGCAGGCATCACCACGCCCATCATCGACACCTCCAAGGTGCC1740              CAAGGAGATGTTCACAGTGGGCTGCTGTGTCTTTACATTCCTCGTGGGACTGTTGTTCGT1800              CCAGCGCTCCGGAAACTACTTTGTCACCATGTTCGATGACTACTCAGCCACGCTGCCACT1860              CACTCTCATCGTCATCCTTGAGAACATCGCTGTGGCCTGGATTTATGGACCCAAGAAGTT1920              CATGCAGGAGCTGACGGAGATGCTGGGCTTCCGCCCCTACCGCTTCTATTTCTACATGTG1980              GAAGTTCGTGTCTCCACTATGCATGGCTGTGCTCACCACAGCCAGCATCATCCAGCTGGG2040              GGTCACGCCCCCGGCCTACAGCGCCTGGATCAAGGAGGAGGCTGCCGAGCGCTACCTGTA2100              TTTCCCCAACTGGCCCATGGCACTCCTGATCACCCTCATCGTCGTGGCGACGCTGCCCAT2160              CCCTGTGGTGTTCGTCCTGCGGCACTTCCACCTGCTCTCTGATGGCTCCAACACCCTCTC2220              CGTGTCCTACAAGAAGGCCCGCATGATGAAGGACATCTCCAACCTGGAGGAGAACGATGA2280              GACCCGCTTCATCCTCAGCAAGGTGCCCAGTGAGGCACCTTCCCCCATGCCCACTCACCG2340              TTCCTATCTGGGGCCCGGCAGCACATCACCCCTGGAGACCAGCTGGAACCCCAATGGACC2400              CTATGGGCGCGGCTACCTGCTGGCCAGCACCCCTGAGTCTGAGCTGTGACCACTGCCCAA2460              GCCCATGCCCGCTCTCCCCCCACCG2485                                                 (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 727 AMINO ACIDS                                                   (B) TYPE: AMINO ACID                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: PROTEIN                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetProLysAsnSerLysValThrGlnArgGluHisSerSerGlu                                 51015                                                                         HisValThrGluSerValAlaAspLeuLeuAlaLeuGluGluPro                                 202530                                                                        ValAspTyrLysGlnSerValLeuAsnValAlaGlyGluAlaGly                                 354045                                                                        GlyLysGlnLysAlaValGluGluGluLeuAspAlaGluAspArg                                 505560                                                                        ProAlaTrpAsnSerLysLeuGlnTyrIleLeuAlaGlnIleGly                                 657075                                                                        PheSerValGlyLeuGlyAsnIleTrpArgPheProTyrLeuCys                                 808590                                                                        GlnLysAsnGlyGlyGlyAlaTyrLysValProTyrLeuValLeu                                 95100105                                                                      LeuIleIleIleGlyIleProLeuPhePheLeuGluLeuAlaVal                                 110115120                                                                     GlyGlnArgIleArgArgGlySerIleGlyValTrpHisTyrIle                                 125130135                                                                     CysProArgLeuGlyGlyIleGlyPheSerSerCysIleValCys                                 140145150                                                                     LeuPheValGlyLeuTyrTyrAsnValIleIleGlyTrpSerIle                                 155160165                                                                     PheTyrPhePheLysSerPheGlnTyrProLeuProTrpSerGlu                                 170175180                                                                     CysProValValArgAsnGluSerValAlaValValGluAlaGlu                                 185190195                                                                     CysGluLysSerSerAlaThrThrTyrPheTrpTyrArgGluAla                                 200205210                                                                     LeuAspIleSerAspSerIleSerGluSerGlyGlyLeuAsnTrp                                 215220225                                                                     LysMetThrLeuCysLeuLeuValValTrpSerIleGlyGlyMet                                 230235240                                                                     AlaValGlyLysGlyIleGlnSerSerGlyLysValMetTyrPhe                                 245250255                                                                     SerSerLeuPheProTyrValValLeuAlaCysPheLeuValArg                                 260265270                                                                     GlyLeuLeuLeuArgGlyAlaValAspGlyIleLeuHisMetPhe                                 275280285                                                                     ThrProLysLeuValLysMetLeuAspProGlnValTrpArgGlu                                 290295300                                                                     ValAlaThrGlnValPhePheGlyLeuGlyLeuGlyPheGlyGly                                 305310315                                                                     ValIleValPheSerSerTyrAsnLysGlnAspAsnAsnCysHis                                 320325330                                                                     PheAspGlyAlaLeuValSerPheIleAsnPhePheThrSerVal                                 335340345                                                                     LeuAlaThrLeuValValPheValValLeuGlyPheLysAlaAsn                                 350355360                                                                     IleMetAsnGluLysCysValValGluAsnAlaGluLysIleLeu                                 365370375                                                                     GlyTyrLeuAsnThrAsnValLeuSerArgAspLeuIleProPro                                 380385390                                                                     HisValAsnPheSerHisLeuThrThrLysAspTyrMetGluMet                                 395400405                                                                     AspAsnValIleMetThrValLysGluAspGlnPheSerAlaLeu                                 410415420                                                                     GlyLeuAspProCysLeuLeuGluAspGluLeuAspLysSerVal                                 425430435                                                                     GlnGlyThrGlyLeuAlaPheIleAlaPheThrGluAlaMetThr                                 440445450                                                                     HisPheProThrSerProPheTrpSerValMetPhePheLeuMet                                 455460465                                                                     LeuIleAsnLeuGlyLeuGlySerMetIleGlyThrMetAlaGly                                 470475480                                                                     IleThrThrProIleIleAspThrSerLysValProLysGluMet                                 485490495                                                                     PheThrValGlyCysCysValPheThrPheLeuValGlyLeuLeu                                 500505510                                                                     PheValGlnArgSerGlyAsnTyrPheValThrMetPheAspAsp                                 515520525                                                                     TyrSerAlaThrLeuProLeuThrLeuIleValIleLeuGluAsn                                 530535540                                                                     IleAlaValAlaTrpIleTyrGlyProLysLysPheMetGlnGlu                                 545550555                                                                     LeuThrGluMetLeuGlyPheArgProTyrArgPheTyrPheTyr                                 560565570                                                                     MetTrpLysPheValSerProLeuCysMetAlaValLeuThrThr                                 575580585                                                                     AlaSerIleIleGlnLeuGlyValThrProProAlaTyrSerAla                                 590595600                                                                     TrpIleLysGluGluAlaAlaGluArgTyrLeuTyrPheProAsn                                 605610615                                                                     TrpProMetAlaLeuLeuIleThrLeuIleValValAlaThrLeu                                 620625630                                                                     ProIleProValValPheValLeuArgHisPheHisLeuLeuSer                                 635640645                                                                     AspGlySerAsnThrLeuSerValSerTyrLysLysAlaArgMet                                 650655660                                                                     MetLysAspIleSerAsnLeuGluGluAsnAspGluThrArgPhe                                 665670675                                                                     IleLeuSerLysValProSerGluAlaProSerProMetProThr                                 680685690                                                                     HisArgSerTyrLeuGlyProGlySerThrSerProLeuGluThr                                 695700705                                                                     SerTrpAsnProAsnGlyProTyrGlyArgGlyTyrLeuLeuAla                                 710715720                                                                     SerThrProGluSerGluLeu                                                         725                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GACTAAAGCTTGGCATCAATGCCGAAGAAC30                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GAACTTCTAGAGCAGTGGTCACAGCTCAG29                                               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GACTAAGATCTGCCACCATGCCGAAGAACAGCAAAGTG38                                      (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 BASE PAIRS                                                     (B) TYPE: NUCLEIC ACID                                                        (C) STRANDEDNESS: SINGLE                                                      (D) TOPOLOGY: LINEAR                                                          (ii) MOLECULE TYPE: Oligonucleotide                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GAACTGATATCGCAGTGGTCACAGCTCAG29                                               __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising apolynucleotide having at least a 95% identity to a member selected fromthe group consisting of:(a) a polynucleotide encoding a polypeptidecomprising amino acids 2 to 727 of SEQ ID NO:2; and (b) the complementof (a).
 2. The isolated polynucleotide of claim 1 wherein said member is(a).
 3. A recombinant vector comprising the polynucleotide of claim 2,wherein said polynucleotide is DNA.
 4. A recombinant host cellcomprising the polynucleotide of claim 2, wherein said polynucleotide isDNA.
 5. The isolated polynucleotide of claim 1 wherein said member is(a) and said polypeptide comprises amino acids 1 to 727 of SEQ ID NO:2.6. The isolated polynucleotide of claim 1, wherein the polynucleotide isDNA.
 7. The isolated polynucleotide of claim 1, comprising apolynucleotide encoding a polypeptide comprising an amino acid sequenceidentical to amino acids 1 to 727 of SEQ ID NO:2.
 8. The isolatedpolynucleotide of claim 1, wherein said polynucleotide is RNA.
 9. Amethod of making a recombinant vector comprising inserting the isolatedpolynucleotide of claim 1 into a vector, wherein said polynucleotide isDNA.
 10. The isolated polynucleotide of claim 1 comprising apolynucleotide comprising nucleotides 271 to 2450 of SEQ ID NO:1. 11.The isolated polynucleotide of claim 1 comprising the polynucleotide ofSEQ ID NO:1.
 12. An isolated polynucleotide comprising a polynucleotidehaving at least 95% identity to a member selected from the groupconsisting of:(a) a polynucleotide encoding the same polypeptide encodedby the human cDNA in ATCC Deposit No. 75713; and (b) the complement of(a).
 13. The isolated polynucleotide of claim 12, wherein the member is(a).
 14. The isolated polynucleotide of claim 12 wherein saidpolynucleotide comprises DNA identical to the coding portion of thehuman cDNA in ATCC Deposit No. 75713 which encodes a mature polypeptide.